1. Field of the Invention
The present invention relates generally to methods for selecting peptide ligands to receptor molecules of interest and for generating and screening large peptide libraries for peptides with desired binding characteristics. The invention also provides methods for optimizing a peptide ligand for improved binding to a receptor and for producing peptide ligands in purified form. The invention therefore relates generally to the field of molecular biology and has application in the field of pharmacology.
2. Description of Related Art
The isolation of ligands that bind biological receptors facilitates the discovery of useful compounds and processes. The ability to synthesize DNA chemically has aided the construction of extremely large collections of nucleic acid and peptide sequences as potential ligands. Recently developed methods allow efficient screening of libraries of biological polymers for desired binding activities (see Pluckthun and Ge, 1991, Angew. Chem. Int. Ed. Enql. 30:296-298). For example, Tuerk and Gold, 1990, Science 249:505-510, report the isolation of RNA molecules with the ability to bind a particular protein and Ellington and Szostak, 1990, Nature 346:818-822, report the isolation of RNA molecules with the ability to bind a dye by alternate rounds of affinity selection and PCR amplification. Thiesen and Bach, 1990, Nucl. Acids Res. 18:3203-3209, used a similar technique to find double-stranded DNA sequences that bind a human transcription factor, while Bock et al., 1992, Nature 355:564-567, and Ellington and Szostak, 1992, Nature 355:850-852, utilized single-stranded DNA libraries.
Efficient peptide screening techniques often utilize physical or logical connections between each peptide and the nucleic acid that encodes each peptide. After rounds of affinity enrichment, such a connection allows identification, usually by amplification and sequencing, of the genetic material encoding interesting peptides. Several phage based systems for screening proteins and polypeptides have been described. The fusion phage approach of Parmley and Smith, 1988, Gene 73:305-318, can be used to screen proteins. Others have described phage based systems in which a peptide is fused to the coat protein of a filamentous phage (see U.S. Pat. No. 5,427,908, filed May 1, 1990; and U.S. Pat. No. 5,723,286, filed Jun. 20, 1990, which is a continuation-in-part of U.S. Pat. No. 5,432,018, filed Jun. 20, 1991, each of which is incorporated herein by reference; see also Scott and Smith, 1990, Science 249:386-390; Devlin et al., 1990, Science 249:404-406; Greenwood et al., 1991, J. Mol. Biol. 220:821-827 and Cwirla et al., 1990, Proc. Natl. Acad. Sci. USA 87:6378-6382; each of which is incorporated herein by reference).
In the latter publications, the authors describe expression of a peptide at the amino terminus of or internal to the pIII or pVIII protein of a filamentous phage. The connection between a peptide and the genetic material that encodes the peptide is established, because the fusion protein is part of the capsid enclosing the phage genomic DNA. Phage encoding peptide ligands for receptors of interest can be isolated from libraries of greater than 10.sup.8 peptides after several rounds of affinity enrichment followed by phage growth. In another random peptide display system, the random peptide library is constructed so that the peptide is expressed as a fusion product with a DNA binding protein (see U.S. Pat. No. 5,270,170, filed Oct. 16, 1991, incorporated herein by reference). The peptide library is constructed so that the DNA binding protein can bind to the recombinant DNA expression vector that encodes the fusion product that contains the peptide of interest.
There remains a need not only for methods of constructing peptide libraries in addition to the methods described above but also for random peptide presentation systems more particularly suited to the optimization of a lead peptide compound. There also remains a need for improved methods and reagents for presenting diverse ligands to a receptor, which methods and reagents force conformational restrictions onto the peptides presented.
The present invention provides random peptide libraries, methods for generating and screening those libraries, and associated reagents with significant advantages over the prior art methods.